Journal:

Article Title: The voltage-gated potassium channel Kv1.3 is highly expressed on inflammatory infiltrates in multiple sclerosis brain

doi: 10.1073/pnas.0501770102

Figure Lengend Snippet: Expression of Kv1.3 on inflammatory cells in MS brain. Paraffin sections were stained by indirect immunoperoxidase for Kv1.3, CD3, CD4, CCR7, and CCR5. Areas used in sectioning were from a white matter plaque. There were many perivascular inflammatory cells that stained positively for CD3 (A), Kv1.3 (B), and CD4 (B Inset) on consecutive sections. (C) Kv1.3 was also localized on inflammatory cells in the white matter parenchyma (Inset reveals membrane polarization of Kv1.3 staining). (D) Consecutive sections through another perivascular infiltrate revealed numerous Kv1.3+ inflammatory cells, which were predominantly CCR7- (E) (Inset reveals rare CCR7 positive staining), and CCR5+ (F). (Scale bar, 50 μmin A and B and 20 μmin C, D, E, and F.)

Article Snippet: The CCR7 antibody was from R & D Systems.

Techniques: Expressing, Staining, Membrane

Journal:

Article Title: The voltage-gated potassium channel Kv1.3 is highly expressed on inflammatory infiltrates in multiple sclerosis brain

doi: 10.1073/pnas.0501770102

Figure Lengend Snippet: Confocal microscopy of Kv1.3 and CD3/CD4 in MS brain tissue. Confocal microscopic images of 20-μm floating sections of postmortem MS brain tissue. (A and B) Parenchymal infiltrate with positive CD3 and punctate Kv1.3 staining in the membrane. (C) A cluster of lymphocytes stained positively for Kv1.3 and CD4, as well as occasional CD8 (Inset). (D) CD3+ cells showed extensive Kv1.3 and CD3 colocalization in the membrane. Immunofluorescent staining of PB-derived T cells concentrated on a slide by cytospin showed membrane patterns of staining of CD4, CD8, and Kv1.3. (E) Day 7-activated naive and TCM are CD4+, CCR7+ (data not shown) and Kv1.3-.(F) Chronically stimulated resting TEM were CD4+, CCR7- (data not shown) and expressed Kv1.3 in the membrane but with minimal colocalization with CD4. (G)TEM that had been recently activated expressed increased amounts of Kv1.3 and demonstrated more colocalization with CD4 similar to the brain tissue lymphocyte in D. (H) Naive CD8+ T cells expressed no Kv1.3 but, when chronically stimulated and activated (I), exhibited intense Kv1.3 expression and colocalization with CD8. (J) Isotype controls using nonspecific rabbit primary antibody followed by usual secondary label and with primary rabbit anti-human and nonspecific labeled mouse anti-rabbit secondary antibody showed no background staining.

Article Snippet: The CCR7 antibody was from R & D Systems.

Techniques: Confocal Microscopy, Staining, Membrane, Derivative Assay, Expressing, Labeling

Journal:

Article Title: The voltage-gated potassium channel Kv1.3 is highly expressed on inflammatory infiltrates in multiple sclerosis brain

doi: 10.1073/pnas.0501770102

Figure Lengend Snippet: Immunofluorescence and electrophysiology of peripheral and CSF T cells. FACS plots revealed staining patterns of CCR7 vs. CD45RA. (A) CD4-gated MS-derived CSF cells revealed a predominantly TCM phenotype immediately ex vivo, but with rapid conversion to short-lived TEM after 7 and 14 days of stimulation using anti-CD3/CD28 coated beads (B and C). By comparison, negatively sorted CSF derived from noninflammatory neurological controls showed CD4+ cells that were predominantly TCM (D) and remained TCM at day 7 despite identical manner of stimulation (E). (F-H) CD4 cells derived from PB were predominantly TCM and naive phenotype (F), and maintained high levels of CCR7 even after 7 and 14 days of stimulation using anti-CD3/CD28 coated beads (G and H). (I) Single cell patch-clamp analysis of MS-derived CSF cells revealed high Kv1.3 channel numbers per cell; mean channel number per cell was 1,082 ± 489 (mean ± SEM, n = 28). Shown for comparison on the right are mean Kv1.3 channel numbers from activated PB T cells from three MS patients (n = 32), and from 10 healthy controls (n = 33). (J and K) Family of currents. The test potential was changed from -60 to 60 mV in 10-mV increments every 30 s (V1/2 = -28 mV). (L) The current in CSF cells exhibited the characteristic use-dependence of Kv1.3 when 200-ms pulses were applied every second to 40 mV. (M and N) Pharmacological blockade with the potent and selective Kv1.3 inhibitor ShK(L5) (14) confirmed that the current is carried by Kv1.3. (O) Representative images of CSF resting (top) or activated (bottom) T cells stained with anti-Kv1.3 and CCR7 Abs. (P) Staining intensities for Kv1.3 and CCR7 in resting and activated CSF T cells. (Q) Z-stack of confocal images through an activated CSF T cell.

Article Snippet: The CCR7 antibody was from R & D Systems.

Techniques: Immunofluorescence, Staining, Derivative Assay, Ex Vivo, Comparison, Patch Clamp

Journal: Virology

Article Title: Integrated proteomics and transcriptomics analyses identify novel cell surface markers of HIV latency

doi: 10.1016/j.virol.2022.06.003

Figure Lengend Snippet: Antibodies used to test enrichment of latently infected cells from mixed cell populations.

Article Snippet: KRAS , Mouse IgG2b , Goat anti-Mouse , PE , Novus Biologicals (NBP2-59413).

Techniques: Infection

Journal: Journal of Biological Chemistry

Article Title: Role of Hypoxia-inducible Factor-1 in Transcriptional Activation of Ceruloplasmin by Iron Deficiency

doi: 10.1074/jbc.m000636200

Figure Lengend Snippet: FIG. 3. Binding of HIF-1 to the HRE of the Cp enhancer (by EMSA). A, Induction of complex formation by HIF-1 agonists. Hep3B cells were exposed for 8 h to 1 mM desferrioxamine (DFO), 1 mM bathophenanthroline sulfate (BPS), or 1% O2 (Hpx.). Nuclear extracts were incubated with 32P-labeled, oligonucleotide 24-mer probes con- taining either the Cp or Epo HRE. Complexes formed were resolved by 5% nondenaturing polyacrylamide gel electrophoresis and visualized by autoradiography. The positions of the putative HIF-1, constitutive (Const.), and nonspecific (NS) complexes are indicated by arrows. B, competitor binding to show specificity of HIF-1 binding to the Cp enhancer HRE. Hep3B cells were treated with 1% O2 for 9 h, and nuclear extracts were prepared as in A. A 10-, 100-, or 1000-fold molar excess of unlabeled, annealed oligonucleotide competitor representing the wild-type (wt) Cp HRE, the mutant (mut) Cp HRE, or the Epo HRE was added to the nuclear extract reaction mixture just prior to addition of radiolabeled Cp HRE probe. The mutated sequence in the Cp HRE is underlined. C, identification of HIF-1 subunits binding to the Cp en- hancer HRE by gel supershift analysis. Hep3B cells were treated with desferrioxamine, and nuclear extracts were prepared as in A. Before subjecting extracts to electrophoresis, the mixtures containing 32P- labeled Cp HRE probe was incubated with 1 ml of anti-HIF-1a, anti- HIF-1b, or both. The supershifted complex is indicated by the open- headed arrow.

Article Snippet: For gel supershift analysis, 1 ml of rabbit monoclonal antibody against HIF-1a or rabbit polyclonal antibody against ARNT/HIF-1b (both from Novus Biologicals, Littleton, CO) was added after the initial 20-min incubation, and the solution was further incubated for 30 min at 4 °C before electrophoresis.

Techniques: Binding Assay, Incubation, Labeling, Polyacrylamide Gel Electrophoresis, Autoradiography, Mutagenesis, Sequencing, Electrophoresis